Nucleic acid probe technology has developed rapidly in recent years as researchers have discovered its value for detection of various diseases, organisms or genetic features which are present in small quantities in a human or animal test sample. The use of probes is based upon the concept of complementarity. DNA has two strands bound together by hydrogen bonds between complementary nucleotides (which are also known as nucleotide pairs). The DNA complex is normally stable, but the strands can be separated (or denatured) by conditions which disrupt the hydrogen bonding. The released single strands will reassociate only with another strand having a complementary sequence of nucleotides. This hybridization process can occur with both strands being in solution or with one of the strands being attached to a solid substrate.
A target nucleic acid sequence in an organism or cell may be only a very small portion of the entire DNA molecule so that it is very difficult to detect its presence using most labeled DNA probes. Much research has been carried out to find ways to detect only a few molecules of a target nucleic acid.
A significant advance in the art is described in U.S. Pat. No. 4,683,195 (Mullis et al), U.S. Pat. No. 4,683,202 (Mullis) and U.S. Pat. No. 4,965,188 (Mullis et al). These patents describe amplification and detection methods wherein primers are hybridized to the strands of a target nucleic acid (considered the templates) in the presence of a nucleotide polymerization agent (such as a DNA polymerase) and deoxyribonucleoside triphosphates. Under specified conditions, the result is the formation of primer extension products as nucleotides are added along the templates from the 3'-end of the primers. These products are then denatured and used as templates for more of the same primers in another extension reaction. When this cycle of denaturation, hybridization and primer extension is carried out a number of times (for example 25 to 30 cycles), the process which is known as "polymerase chain reaction" (or PCR) exponentially increases the original amount of target nucleic acid so that it is readily detected.
Once the target nucleic acid has been sufficiently amplified, various detection procedures can be used to detect it, as noted in the cited patents.
Various devices have been designed for hybridization assays whereby a target nucleic acid is insolubilized prior to detection using appropriate detection probes. For example, nitrocellulose filters and other planar, solid supports have been used for this purpose as described for example, in U.S. Pat. No. 4,925,785 (Wang et al).
One technique used to attach probes or other reagents is merely to dry them down onto the solid support, as described for example in EP-A-0 381 501 (published Aug. 8, 1990). More recently, probes attached to polymeric particles are fused into a solid support formed of another polymer which can be softened by heat (see U.S. Ser. No. 639,454, filed Jan. 10, 1991 by Zander et al).
While providing improvement over prior techniques, immobilization of capture reagents (such as capture probes) by drying or fusing has limitations. Adhesion of the reagents is poor. Particularly in assays whereby reagents in solution are washed over the immobilized capture probes, some of the probes are dislodged and potential signal is lost when the probes are washed away. Moreover, fusion of capture probes to a solid support requires high temperatures and dangerous equipment which tend to reduce the ability of the probes to capture a target nucleic acid.
It would be desirable to avoid the problems associated with known means for immobilizing capture probes while providing highly sensitive assays with minimal loss in signal associated with loss of capture probe.